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An Intronic Variant of CHD7 Identied in Autism Patients Interferes With Neuronal Differentiation and Development Ran Zhang Institute of Neuroscience Chinese Academy of Sciences Hui He Institute of Neuroscience Chinese Academy of Sciences Bo Yuan Institute of Neuroscience Chinese Academy of Sciences Ziyan Wu Institute of Neuroscience Chinese Academy of Sciences Xiuzhen Wang Institute of Neuroscience Chinese Academy of Sciences Yasong Du Shanghai Mental Health Center Yuejun Chen Institute of Neuroscience Chinese Academy of Sciences Zilong Qiu ( [email protected] ) Institute of Neuroscience, Chinese Academy of Sciences https://orcid.org/0000-0003-4286-3288 Research Keywords: Autism, CHD7, Intronic variant, Inherited variant Posted Date: August 17th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-58118/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Neuroscience Bulletin on May 4th, 2021. See the published version at https://doi.org/10.1007/s12264-021-00685-w. Page 1/27 Abstract Background: Genetic compositions play critical roles in pathogenesis of autism spectrum disorders (ASD). Inherited and de novo intronic variants are often seen in patients with ASD. However, the biological signicance of intronic variants are dicult to address. Here, we identied a recurrent inherited intronic variant in the CHD7 gene among Chinese ASD cohort, which is specically enriched in East Asian populations. CHD7 has been implicated in numerous developmental disorders including CHARGE syndrome and ASD. Here, we use differentiated human embryonic stem cells to investigate whether the ASD-associated CHD7 intronic variant may affect neural development. Methods: 167 ASD probands and their parents performed whole-exome sequencing and a recurrent inherited intronic variant was selected for functional research. We established human embryonic stem cells carrying this intronic variant using CRISPR/Cas methods and differentiated to neural progenitor cells and neurons for analysis. Results: The level of CHD7 mRNA signicantly decreased in cell lines carrying homozygous mutants compared to control. Upon differentiation towards the forebrain neuronal lineage, neural cells carrying the CHD7 intronic variant exhibited developmental delay in neuronal differentiation and maturation. Importantly, TBR1, a gene also implicated in ASD, signicantly increased in neurons carrying the CHD7 intronic variant, suggesting the intrinsic relevance among ASD genes. Furthermore, the morphological defects found in neurons carrying CHD7 intronic mutations could be rescued by knocking down TBR1, indicating that TBR1 may be responsible for defects in CHD7-related disorders. Finally, the CHD7 intronic variant generates three abnormal forms of transcripts through alternative splicing, which all exhibited loss-of-function manners in functional assays. Limitations: The probands with CHD7 intronic variant were heterozygous while both cell lines established were homozygous, so the phenotype of the disease could not be completely simulated. Conclusions: Our study provides the crucial evidences for supporting the notion that intronic variant site of CHD7 is a potentially autism susceptible site, shedding new light on identifying functions of intronic variants in autism genetic studies. Background Autism spectrum disorder (ASD) is known as a specic group of neurodevelopmental disorders characterized by impairments in social interaction and stereotyped behaviors (Kanner 1943). Although both genetic and environmental factors may contribute to the autistic symptoms, recent genome-wide analysis indicate that the genetic composition is the dominant cause for ASD (Ronald and Hoekstra 2011, Sandin, Lichtenstein et al. 2014, Tick, Bolton et al. 2016). The genetic architecture of ASD is highly heterogeneous as over a hundred of risk genes have been found (Huguet and Bourgeron 2016, Satterstrom, Kosmicki et al. 2020). Analysis of de novo and inherited rare variation linked to ASD has Page 2/27 identied convergent functional themes, such as neuronal development and axon guidance, signaling pathways, and chromatin and transcription regulation (Pinto, Delaby et al. 2014, Bourgeron 2015). Due to the genetic heterogeneity, one strategy is to perform genome sequencing for a large number of ASD families and identify risk genes more comprehensively. Benet from the increasing resolution and reducing cost of DNA sequencing technology such as karyotype, microarrays, whole-exome sequencing and whole-genome sequencing, nearly 20% of ASD cases can be explained by identiable genetic causes, while the rest remained unclear. Till now, most efforts were focused on rare deleterious de novo single- nucleotide variants (likely gene-disrupting variants-LGD variants) on coding regions or splicing sites. However, multiple common inherited variants are clearly suspectable factors for ASD (Gaugler, Klei et al. 2014). Since inherited variants are also present in unaffected parents, identifying the causal relationship between inherited variations and ASD pathogenesis would require neurobiological evidences besides genetic evidences (Jiang, Yuen et al. 2013, Guo, Wang et al. 2018). The current largest genetic sequencing projects for ASD cohorts are from Europe and the United States, which are mostly composed of Caucasians, Latino, Ashkenazi Jewish and African Americans populations etc. Due to the genetic geographical difference between Caucasian and Asians, the potential genetic causes for Asians population may be incomplete overlap with those in the western world. Considering the vast population of China, there is no doubt a large population of ASD in Chinese populations. Identication of the ASD risk genes based on Chinese population not only critical to provide precise genetic diagnosis and specic interventions for Chinese ASD patients, but also provides the important missing piece for a comprehensive and in-depth understanding of autism. In this study, we performed the whole-exome sequencing for 167 Chinese ASD proband along with their parents. We identied an intronic inherited variation of chromodomain-helicase-DNA-binding protein 7 (CHD7) (ch8-61757392-C-T), which exclusively appears in East Asian populations by 0.39% in the gnomAD database, while enriched by 3.6% in our Chinese ASD cohort (6/167), suggesting that this intronic inherited variant may function as a susceptibility variant for autism. The CHD7 protein belongs to the CHD family of chromatin remodelers and catalyses the translocation of nucleosomes along DNA in chromatin (Petty and Pillus 2013). Mutations of the CHD7 gene are the major cause of the CHARGE syndrome, which is characterized by coloboma of the eye, heart defects, atresia of the choanae, retardation of growth and development, genital abnormalities, and ear abnormalities (Vissers, van Ravenswaaij et al. 2004). Children with the CHARGE syndrome frequently exhibit the autistic-like decits on vocalization, social responsiveness, and repetitive behaviors, suggesting that CHD7 has a more direct impact on autism (Hartshorne, Grialou et al. 2005). To investigate whether this intronic variation may affect the proper expression of the CHD7, we constructed two homozygous point mutant cell lines using human H9 embryonic stem cells (hESCs). We found that the relative mRNA amount of CHD7 is downregulated after introducing of the intronic point mutation in both cell lines. The differentiation of the neural precursor cells (NPCs) carrying intronic mutations appears to delay, comparing to wild-type NPCs. Importantly, when NPCs carrying intronic Page 3/27 mutations differentiated into neurons, the dendrite length and branch number are signicantly lower than neurons which do not carrying mutations, which could be rescued by knockdown TBR1. Interestingly, we found that the intronic mutation affect alternative splicing of CHD7 mRNA by introducing three kinds of non-canonical transcripts. We further showed that the three non-canonical transcripts are loss-of- functions mutants in mouse culture neurons. In summary, we identify an intronic inherited variation in the CHD7 gene enrich in the Chinese ASD cohorts. The intronic variation leads to downregulation of CHD7 mRNA and non-canonical alternative splicing process, affecting processes related to neurodevelopment which could be rescued by knockdown its target gene TBR1. This study demonstrates that the intronic inherited variation of CHD7 is a potential suspectable factor for autism specically in Chinese ASD cohorts, opening up a new venue for studying the complex genetic causes of autism. Methods Subjects A total of 168 core families with probands diagnosed with ASD were recruited from the outpatient of Department of the Child and Adolescent Psychiatry, Shanghai Mental Health Center. All patients were diagnosed on the basis of the fth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-V). All patients were Han Chinese and their age was ranged from 2 to 18 years. Human H9 embryonic stem cells (hESCs) and intronic mutation (8-61757392-C-T) hESCs hESCs (H9, line WA09 (WiCell), passages 20-40) were cultured on a feeder layer of irradiated mouse embryonic broblasts (MEFs) in a humidied incubator with 5%CO2 at 37℃. The hESC medium containing DMEF/F12, 20% Knock Serum Replacement (Life Technologies), 0.1mM beta- mercaptoethanol (Sigma), 1% (v/v) Non-Eessential Amino Acids (NEAA, Life Technologies), 0.5% (v/v) GlutaMAX (Life Technologies).
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